Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein. A deep-etch and immunolocalization study of ultrarapidly frozen tissue.

Abstract

The intima from aortas of normal New Zealand White rabbits was studied 2 hours after infusion of 320 mg human low density lipoprotein (LDL), resulting in a plasma concentration of at least five times and maximally 20 times the values found in normal rabbit serum. The following techniques were used: 1) ultrarapid freezing without chemical fixation, followed by freeze-etching; 2) immunofluorescence microscopy; and 3) postembedding immunogold-labeling electron microscopy. In the latter two methods MB47, a murine monoclonal antibody against human apolipoprotein B, was used as the primary antibody. Control rabbits were infused with the same volume of buffer only. Rotary-shadowed replicas of samples from the LDL-injected rabbits showed the deposition of lipidlike particles in the subendothelial-intimal space that were the size of the injected LDL (23 nm). In focal areas of the intima, groups of 23-nm-sized lipidlike particles and larger lipidlike structures were found enmeshed in the extracellular matrix. Control replicas were essentially free of lipid deposition. Immunofluorescence microscopy of frozen aortic cross sections showed an overall increase in apolipoprotein B in the intima of the LDL-injected rabbits. The presence of apolipoprotein B in the intima was also confirmed by immunoelectron microscopy. These in vivo results show that clustering of LDL-sized particles occurs in the intima within 2 hours of excessive LDL uptake. It also demonstrates the interaction of these LDL-sized particles with the filaments of the extracellular matrix. The clustering of the LDL-sized particles supports the possibility that LDL selfaggregation may occur in vivo and that components of the extracellular matrix are involved in this process. {Arteriosclerosis and Thrombosis 1991;ll:1795-1805)

Implications of Early Structural-Functional Changes in the Endothelium for Vascular Disease

“…This observation was further confirmed on Watanabe heritable hyperlipemic rabbits (WHHL) and cholesterol-fed rabbits 29,30,31 and in intimal thickening of human aorta. 32,33 Although the exact location and the mechanism(s) of MRL formation are not completely understood, one can safely assume that the process may take place either on LDL interaction with ECs, or during transcytosis or within the subendothelial space by extended interaction with matrix components and cells.…”

“…37,38 , presumably slightly modified Lp that have increased propensity to bind PG. Several lines of evidence support the hypothesis that MRL may be the key event and first change occurring in atherogenesis: (1) although in normal artery wall there is constant traffic of native plasma Lp through PG-rich matrix, no retention or modification of Lp occur; (2) within minutes to hours after the onset of hyperlipidemia, the first identifiable process (before monocyte diapedesis or foam cell formation) is the accumulation of subendothelial MRL; 25,31 (3) in vitro, human LDL autoxidation (in the absence of PG) generates MRL structurally and chemically similar to those detected in vivo at the inception of atherogenesis; 39 (4) matrix PG have increased affinity for LDL isolated from patients with myocardial infarction; 37 and (5) in vitro, MRL have chemotactic properties for monocytes (unpublished data).…”

Sphingolipids in Atherosclerosis and Vascular Biology

“…71 Several mechanisms have been implicated in the initiation of early atherogenesis. Some of these processes are LDL oxidation, 91 lipoprotein retention and aggregation, 92,93 endothelial cell dysfunction, the expression of adhesion molecules, 94 monocyte/macrophage adhesion to the endothelial cells, foam cell formation, and smooth muscle cell phenotypic changes involving proliferation as well as migration. 95 Recent studies have implicated several sphingolipids to be involved in the phenomenon above.…”

Sphingomyelinase, an Enzyme Implicated in Atherogenesis, Is Present in Atherosclerotic Lesions and Binds to Specific Components of the Subendothelial Extracellular Matrix

“…Our laboratory has focused on LDL aggregation, which is a prominent event in the subendothelial space during atherogenesis, [27][28][29] and on the potential role of S-SMase in mediating this aggregation. 9,11,30 Arterial-wall macrophages loaded with large amounts of cholesteryl ester (macrophage foam cells) are thought to play a major role throughout atherogenesis.…”